Differences in inducibility of the latent HIV reservoir in perinatal and adult infection

Abstract

The HIV latent reservoir in resting memory CD4+ T cells precludes cure. Therapeutics to reactivate and eliminate this reservoir are in clinical trials in adults, but not yet in pediatric populations. We determined, ex vivo, the inducibility of the latent reservoir in perinatal infection as compared with adult infections using the Tat/rev induced limiting dilution assay (TILDA), in which a single round (12 hours) of CD4+ T cell stimulation with PMA/ionomycin maximally activates T cells and leads to proviral expression with multiply spliced HIV RNA production. Markers of immune activation and exhaustion were measured to assess interactions with inducibility. Although rates of T cell activation with PMA/ionomycin were similar, the latent reservoir in perinatal infection was slower to reactivate and of lower magnitude compared with adult infection, independent of proviral load. An enhanced TILDA with the addition of phytohemagglutin and a duration of 18 hours augmented proviral expression in perinatal but not adult infection. The baseline HLA-DR+CD4+ T cell level was significantly lower in perinatal compared with adult infections, but not correlated with induced reservoir size. These data support the hypothesis that there are differences in kinetics of latency reversal and baseline immune activation in perinatal compared with adult infections, with implications for latency reversal strategies toward reservoir clearance and remission.

Keywords: AIDS/HIV; Immunology; Molecular biology; T cells.

Figure 1. The viral reservoir in perinatal infection is more resistant to reactivation than in adult infection.

(A) Proviral load as quantified by both ddPCR measuring GAGLTR only and double-positive intact droplets as measured by IPDA (23). Perinatally infected samples are shown in blue (subtype B) and red (non–subtype B). Adult samples are labeled in orange; open symbols indicate samples that were undetectable by the specified assay. (B) Size of the inducible reservoir quantified as multiply spliced HIV RNA–producing units per million CD4⁺ T cells (msRUPM) in participants with perinatal (n = 11) and adult (n = 10) infection as measured by standard (circles) and enhanced TILDA (squares). (C) Fold change in the size of the inducible reservoir with the enhanced stimulation condition in perinatal and adult infections. Bars represent median and IQR. The significance of differences between populations or stimulation conditions was calculated using nonparametric 2-tailed unpaired Mann-Whitney U tests and paired Wilcoxon’s tests.

Figure 2. A small fraction of proviruses retained in the latent reservoir in perinatal and adult HIV infection are induced to produce multiply-spliced HIV RNA transcripts.

(A) Percentage of proviruses in CD4⁺ T cells that were induced under standard and enhanced TILDA conditions in perinatal (n = 11, subtype B in blue and non–subtype B in red) and adult (n = 10, in orange) infections, normalized to GAGLTR copies per calculated CD4⁺ T cells. (B) Percentage of proviruses in CD4⁺ T cells that were induced under standard and enhanced TILDA conditions in perinatal (n = 7) and adult (n = 4) infections, normalized to double-positive intact copies per calculated CD4⁺ T cells as detected by IPDA. Five samples from perinatal infections were excluded from the IPDA analysis due to undetectable double-positive droplets, and thus could be used to normalize the TILDA. Open symbols represent individuals with nondetectable inducible reservoir. Bars represent median and IQR. The significance of the difference in percent induced provirus was determined using nonparametric 2-tailed unpaired Mann-Whitney U tests and paired Wilcoxon’s tests.

Figure 3. Plot of enhanced TILDA against ddPCR proviral DNA assays.

Multiply spliced RNA producing units per million PBMCs (msRUPM) plotted against GAGLTR copies per million PBMCs in (A) adult (n = 10, orange) individuals and (B) perinatally infected (n = 11, subtype B in blue and non–subtype B in red) individuals. Open data points represent individuals with undetectable msRUPM. (C) msRUPM plotted against double-positive intact proviruses per million PBMCs using IPDA. There was no correlation between TILDA and total DNA ddPCR, or between TILDA and IPDA. Spearman’s rank correlation coefficients and the corresponding P values are shown in the graphs.

Figure 4. Percentage of CD4⁺ T cells displaying activation markers.

(A) CD69, (B) CD25, and (C) HLA-DR expression during baseline. Standard and enhanced TILDA stimulation conditions in perinatal (left, n = 7) and adult (right, n = 4) infections.

Figure 5. Baseline levels of activation markers.

Comparison of the activation markers (A) CD69, (B) CD25, (C) HLA-DR and the exhaustion markers (D) PD-1, (E) TIM3, and (F) TIGIT between perinatal (n = 7) and adult (n = 4) infections as measured by flow cytometry. Bars represent median and IQRs. The tests for the differences in percent cells with the shown surface markers was conducted using a nonparametric 2-tailed unpaired Mann-Whitney U test.

Figure 6. Schematic of experimental conditions summarizing CD4⁺ T cell selection and stimulation and subsequent assay conditions.

Cryopreserved PBMCs are thawed and enriched for CD4⁺ T cells by negative bead enrichment. Cells are subsequently aliquoted at 2 million cells per 1 mL of medium and rested for 2–6 hours. Following the rest period, each sample is distributed into individual wells as (i) unstimulated, for subsequent flow studies for baseline measurements, (ii) stimulated, using PMA and ionomycin for 12 hours (standard TILDA), or (iii) stimulated, with PMA, PHA, and ionomycin for 18 hours (enhanced TILDA). After stimulation, cells are washed and either fixed and stained for flow cytometry, or plated in a limiting dilution scheme for direct lysis and RT-qPCR followed by qPCR as previously published.